Dynamics of endomembrane docking and fusion

内膜系统中的对接和融合事件由Rab GTP酶调节和执行， SNARE蛋白及其辅助因子。关于这些蛋白质及其 交互.然而，导致融合的分子事件对于任何细胞内的细胞内融合都没有被详细地理解。 运输步骤。解决单个事件而不是事件的聚合行为的能力 人口的增长促进了生物学许多领域的重大进步。我们相信机械论 如果我们能够追踪和扰动膜的结构， 在单个对接和融合事件的水平上的系留、对接和融合的子反应。在这 建议我们联合收割机直接攻击的问题，监测单一事件与生物化学和遗传 这些研究将同时表征关键分子并产生新的试剂和探针， 单一事件工作。我们的实验系统，酵母液泡，提供了许多实验优势 对于这些研究，包括它的大小，这是大到足以方便光学显微镜，遗传和 芽殖酵母的基因组工具包，以及无细胞融合系统，在其实验中是无与伦比的。 灵活性和现有的知识基础。这些实验将建立在-并大大扩展- 这是Pi在William Wickner博士的小组中进行博士后工作期间开发的方法。微制造 技术和低光定量荧光显微镜将;与无细胞液泡相结合 融合系统，以监测和干扰个别酵母液泡，因为他们系绳，码头，和融合。我们将重点 我们在Ypt 7 p，空泡Rab，其Vps-C/HOPS效应复合物和Vam 7 p，可溶性SNARE上的努力。 这些蛋白质在整个拴系融合序列中具有关键功能，并且许多蛋白质在整个拴系融合序列中具有关键功能。 功能和物理相互作用将它们联系起来。我们要解决的生物学问题很简单。 GTP在Rab Ypt 7 p上的水解和交换是否影响束缚的力或可逆性，或 对接接头组装动力学（目标1）？SNARE Vam 7 p在执行其 对接和聚变的各种功能（目标2）？Vps-C复合体是如何组织的， 该复合物响应Ypt 7 p结合或其它对接事件而经历结构重排， 融合（目标3）？Ypt 7 p是否“知道”何时发生了系留、对接或融合（目标4）？

Docking and fusion events in the endomembrane system are regulated and executed by Rab GTPases, SNARE proteins, and their cofactors. A great deal has been learned about these proteins and their interactions. However, the molecular events leading to fusion are not understood in detail for any intracellular transport step. The ability to resolve individual events rather than the aggregate behavior of event populations has potentiated major advances in many areas of biology. We believe that the mechanistic dissection of membrane docking and fusion would be greatly facilitated if we could trace and perturb the subreactions of tethering, docking, and fusion at the level of individual docking and fusion events. In this proposal we combine direct attacks on the problem of monitoring single events with biochemical and genetic studies that will simultaneously characterize critical molecules and yield new reagents and probes for the single-event work. Our experimental system, the yeast vacuole, offers numerous experimental advantages for these studies including its size, which is large enough to facilitate light microscopy, the genetic and genomic toolkit of budding yeast, and a cell-free fusion system that is unsurpassed in its experimental flexibility and existing knowledge base. These experiments will build on- andsubstantially extend - approaches developed during the Pi's postdoctoral work in Dr. William Wickner's group. Microfabrication techniques and low-light, quantitative fluorescence microscopy will be; combined with the cell-free vacuole fusion system to monitor and perturb individual yeast vacuoles as they tether, dock, and fuse. We will focus our efforts on Ypt7p, the vacuole Rab, its Vps-C/HOPS effector complex, and Vam7p, a soluble SNARE. These proteins have critical functions over the entire span of the tethering-to fusion sequence, and many functional and physical interactions link them. The biological questions that we address are straightforward. Does GTP hydrolysis and exchange on the Rab Ypt7p influence the force or reversibility of tethering, or the dynamics of docking junction assembly (Aim 1)? What does the SNARE Vam7p touch as it executes its various functions in docking and fusion (Aim 2)? How is the Vps-C complex organized, and do subunits of this complex undergo structural rearrangements in response to Ypt7p-binding or other events of docking or fusion (Aim 3)? Does Ypt7p "know" when tethering, docking, or fusion have occurred (Aim 4)?